Chitin is a polymer of beta-1-4-N-acetyl-D-glucosamine found in fungal cell walls and crustacean shells. A common source of chitin consists of shells of marine invertebrates such as crabs and shrimps. Waste from industrial microbiological plants using fermentation methods with fungal organisms is another source of chitin.
Chitin is insoluble in water and most solvents. Its high hydrophobicity is the main reason of poor susceptibility to enzymatic hydrolysis. Enzymes that hydrolyse chitin--chitinases--have low specific activities and hydrolyse the polymer very slowly.
Chitin can be deacetylated partially or totally. Such a deacetylated polymer is called chitosan. In nature, chitosan is present only in cell walls of Zygomycetes, a group of phytopathogenic fungi. In contrast with chitin, chitosan is much less hydrophobic and is soluble in diluted acids (for instance in diluted HCl or in acetic acid). Because of its significant content of free amino groups, chitosan has a markedly cationic character and has a positive charge at most pHs. Enzymatic hydrolysis of chitosan is much easier than in the case of chitin.
Chitin deacetylation towards chitosan can be obtained by various methods. The most used method is that of alkaline treatment (Horowitz, S. T. et al., 1957). With this method, around 80% of deacetylation can be achieved without significant decrease of molecular weight. A more intense deacetylation cannot be obtained by this method without a simultaneous uncontrolled decrease of the degree of polymerization. A more promising method, but still under development, is deacetylation by a thermo-mechano-chemical treatment (Pelletier et al., 1990). This method allows a more careful control of the various characteristics of the final product (average degree of polymerisation and of deacetylation). Finally, a third method (Domard and Rinaudo, 1983) allows to obtain a totally deacetylated product. However, the cost of chemicals used in this method as well as their toxicity will probably limit the usefulness of this method to the laboratory scale.
High-molecular weight chitosan has many potential applications (reviewed by Sandford, 1989). Some applications are however typical of medium- or low-molecular weight chitosan (oligomeric chitosan). They include its exploitation as an antifungal agent; a seed coating for improving crop yield; an elicitor of anti-pathogenic natural reactions in plants; a hypocholesterolemic agent in animals; an accelerator of lactic acid bacteria breeding; and a moisture-retaining agent for lotions, hair tonics and other cosmetics. Furthermore, the anti-tumoral and immunostimulating properties of low-molecular weight chitosan are under active investigation in various laboratories and could lead to new important applications.
Hydrolysis of high-molecular weight chitosan, giving medium- or low-molecular weight chitosan can be achieved essentially by two methods. The chemical hydrolysis with strong acids (HCl or HF) is efficient but gives little possibility to control efficiently the molecular weight of the final product. The other method is enzymic hydrolysis with chitosanase. This enzyme hydrolyses beta-1,4-D-glucosaminic linkages of chitosan, reducing progressively the molecular weight of the polymer and giving low molecular weight oligomers as final hydrolysis product.
Reviewing the existing scientific and technical literature, we have found that the production of enzymes suitable for providing medium- or low-molecular weight chitosans through enzymatic treatment is often quite low in naturally occurring microorganisms and has to be enhanced in order to be commercially feasible.
Thus by molecular cloning and overexpression of a gene coding for chitosanase, improved production of this enzyme has been attained and found to be of value for chitosan hydrolysis.
The techniques of recombinant gene technology (gene cloning) are known and widely used. However, as yet, there is no known example of cloning of a chitosanase gene.
As a means for the production of an enzymic protein which: 1) exhibits a hydrolase activity, essentially a chitosanase activity; 2) is overproduced and present in a supernatant during the growth of a recombinant Streptomyces lividans microorganism, the present invention provided a gene system coding for such an enzyme.
In the treatment of chitosan with the chitosanase that is present in the culture fluid in which a suitable microorganism is grown, the hydrolysis of beta-1,4-D-glucosamic linkages will lead to a progressive reduction of the molecular weight of chitosan chains, decreasing chitosan viscosity and increasing its solubility in water. Various chitosan-related biological properties (immunostimulating activity in animals; elicitor activity of anti-pathogenic reactions in plants) are highly dependent of the average molecular weight.